Bottom Line:
Unexplainably, monoclonal antibodies (MAbs) with virus-neutralizing activity map to these residues.The absence of any of these proteins abolishes the entry process.Our study supports the concept that gB is the HSV fusogen and its activity is regulated by gH/gL.

Affiliation: Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

ABSTRACT

Unlabelled: Herpesvirus entry requires the viral glycoprotein triad of gB and gH/gL to carry out fusion between the virion envelope and a cellular membrane in order to release the nucleocapsid into the target cell. Herpes simplex virus (HSV) also requires glycoprotein gD to initiate the fusion cascade by binding a cell receptor such as nectin 1 or herpesvirus entry mediator (HVEM). While the structure of gB is that of a class III fusion protein, gH/gL has no features that resemble other viral fusion proteins. Instead, it is suggested that gH/gL acts as a regulator of gB. The crystal structure of HSV-2 gH/gL was obtained with a functional protein that had a deletion of 28 residues at the gH N terminus (gHΔ48/gL). Unexplainably, monoclonal antibodies (MAbs) with virus-neutralizing activity map to these residues. To reconcile these two disparate observations, we studied the ability of gHΔ48/gL to regulate fusion. Here, we show that the protein induces low (constitutive) levels of fusion by gB in the absence of gD and/or receptor. However, when gD and receptor are present, this mutant functions as well as does wild-type (wt) gH/gL for fusion. We propose that gHΔ48/gL has an intermediate structure on the pathway leading to full regulatory activation. We suggest that a key step in the pathway of fusion is the conversion of gH/gL to an activated state by receptor-bound gD; this activated gH/gL resembles gHΔ48/gL.

Importance: Herpes simplex viruses (HSVs) cause many human diseases, from mild cold sores to lethal neonatal herpes. As an enveloped virus, HSV must fuse its membrane with a host membrane in order for replication to take place. The virus uses four glycoproteins for this process, gD, gB, and gH/gL, and either of two cell receptors, herpesvirus entry mediator (HVEM) and nectin 1. Although the virus can enter the cell by direct fusion at the plasma membrane or via endocytosis, the same four glycoproteins are involved. The absence of any of these proteins abolishes the entry process. Here, we show that a mutant form of gH/gL, gHΔ48/gL, can induce fusion of gB-expressing cells in the absence of gD and a gD receptor. Our study supports the concept that gB is the HSV fusogen and its activity is regulated by gH/gL.

fig2: N-terminal gH/gL truncations work at levels comparable to those of wild-type gH2/gL2. C10 cells were transfected with full-length gB (A) and gH/gL: wt gH2/gL2 (B), gHΔ29/gL2 (C), or gHΔ48/gL2 (D). Fusion was triggered with soluble gD306. C10 cells transfected with gB were triggered with a mixture of gD306 (E) and either soluble wt gH2/gL2 (F), truncated gHΔ29t/gL2 (G), or truncated gHΔ48t/gL2 (H). gHΔ48/gL2 is an activated form of gH/gL. C10 cells were transfected with full-length gB, gL, and wild-type gH2 (I), gHΔ29 (J), or gHΔ48 plasmids (K). In a parallel experiment, cells were transfected with gB plasmid only; at 6 h posttransfection, 250 µg/ml of soluble gH2t/gL2 (L), gHΔ29t/gL2 (M), or gHΔ48t/gL2 (N) was added. All coverslips were examined by immunofluorescence with gB MAbs (red). Nuclei were stained with propidium iodide and artificially colored gray using Volocity software. Syncytia are outlined with white dotted lines. N, number of syncytia from a coverslip multiplied by the average number of nuclei per syncytia.

Mentions:
We first compared the abilities of membrane-bound gH2Δ29/gL2, gH2Δ48/gL2, and wild-type (wt) gH2/gL2 to induce fusion of gB-expressing cells upon the addition of soluble gD (Fig. 2). As a control, gH2/gL2 was omitted and no syncytia were detected (Fig. 2A). Next, B78C10 (C10) cells expressing nectin 1 were transfected with plasmids expressing full-length gB, gL2, and either wt gH2 (Fig. 2B), gH2Δ29 (Fig. 2C), or gH2Δ48 (Fig. 2D). In each case, soluble gD2306t was then added to initiate fusion (for easier identification, syncytia were outlined with a white dotted line) (10). As previously shown (23), each form of gH2/gL2 induced fusion in the presence of gD and the fusion activities in each case were approximately the same. The total number of fusion events (the number of syncytia multiplied by the number of nuclei in a syncytium) ranged from 3,480 to 3,960 per coverslip (Fig. 2B to D).

fig2: N-terminal gH/gL truncations work at levels comparable to those of wild-type gH2/gL2. C10 cells were transfected with full-length gB (A) and gH/gL: wt gH2/gL2 (B), gHΔ29/gL2 (C), or gHΔ48/gL2 (D). Fusion was triggered with soluble gD306. C10 cells transfected with gB were triggered with a mixture of gD306 (E) and either soluble wt gH2/gL2 (F), truncated gHΔ29t/gL2 (G), or truncated gHΔ48t/gL2 (H). gHΔ48/gL2 is an activated form of gH/gL. C10 cells were transfected with full-length gB, gL, and wild-type gH2 (I), gHΔ29 (J), or gHΔ48 plasmids (K). In a parallel experiment, cells were transfected with gB plasmid only; at 6 h posttransfection, 250 µg/ml of soluble gH2t/gL2 (L), gHΔ29t/gL2 (M), or gHΔ48t/gL2 (N) was added. All coverslips were examined by immunofluorescence with gB MAbs (red). Nuclei were stained with propidium iodide and artificially colored gray using Volocity software. Syncytia are outlined with white dotted lines. N, number of syncytia from a coverslip multiplied by the average number of nuclei per syncytia.

Mentions:
We first compared the abilities of membrane-bound gH2Δ29/gL2, gH2Δ48/gL2, and wild-type (wt) gH2/gL2 to induce fusion of gB-expressing cells upon the addition of soluble gD (Fig. 2). As a control, gH2/gL2 was omitted and no syncytia were detected (Fig. 2A). Next, B78C10 (C10) cells expressing nectin 1 were transfected with plasmids expressing full-length gB, gL2, and either wt gH2 (Fig. 2B), gH2Δ29 (Fig. 2C), or gH2Δ48 (Fig. 2D). In each case, soluble gD2306t was then added to initiate fusion (for easier identification, syncytia were outlined with a white dotted line) (10). As previously shown (23), each form of gH2/gL2 induced fusion in the presence of gD and the fusion activities in each case were approximately the same. The total number of fusion events (the number of syncytia multiplied by the number of nuclei in a syncytium) ranged from 3,480 to 3,960 per coverslip (Fig. 2B to D).

Bottom Line:
Unexplainably, monoclonal antibodies (MAbs) with virus-neutralizing activity map to these residues.The absence of any of these proteins abolishes the entry process.Our study supports the concept that gB is the HSV fusogen and its activity is regulated by gH/gL.

Affiliation:
Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

ABSTRACT

Unlabelled: Herpesvirus entry requires the viral glycoprotein triad of gB and gH/gL to carry out fusion between the virion envelope and a cellular membrane in order to release the nucleocapsid into the target cell. Herpes simplex virus (HSV) also requires glycoprotein gD to initiate the fusion cascade by binding a cell receptor such as nectin 1 or herpesvirus entry mediator (HVEM). While the structure of gB is that of a class III fusion protein, gH/gL has no features that resemble other viral fusion proteins. Instead, it is suggested that gH/gL acts as a regulator of gB. The crystal structure of HSV-2 gH/gL was obtained with a functional protein that had a deletion of 28 residues at the gH N terminus (gHΔ48/gL). Unexplainably, monoclonal antibodies (MAbs) with virus-neutralizing activity map to these residues. To reconcile these two disparate observations, we studied the ability of gHΔ48/gL to regulate fusion. Here, we show that the protein induces low (constitutive) levels of fusion by gB in the absence of gD and/or receptor. However, when gD and receptor are present, this mutant functions as well as does wild-type (wt) gH/gL for fusion. We propose that gHΔ48/gL has an intermediate structure on the pathway leading to full regulatory activation. We suggest that a key step in the pathway of fusion is the conversion of gH/gL to an activated state by receptor-bound gD; this activated gH/gL resembles gHΔ48/gL.

Importance: Herpes simplex viruses (HSVs) cause many human diseases, from mild cold sores to lethal neonatal herpes. As an enveloped virus, HSV must fuse its membrane with a host membrane in order for replication to take place. The virus uses four glycoproteins for this process, gD, gB, and gH/gL, and either of two cell receptors, herpesvirus entry mediator (HVEM) and nectin 1. Although the virus can enter the cell by direct fusion at the plasma membrane or via endocytosis, the same four glycoproteins are involved. The absence of any of these proteins abolishes the entry process. Here, we show that a mutant form of gH/gL, gHΔ48/gL, can induce fusion of gB-expressing cells in the absence of gD and a gD receptor. Our study supports the concept that gB is the HSV fusogen and its activity is regulated by gH/gL.